Diseases which are inherited from an individual's parents are known as genetic diseases. At least 1,500 distinguishable human diseases are already known to be genetically determined (McKusick, V. A., Mendelian Inheritance in Man (Johns Hopkins Press, Baltimore, 3rd. Ed., 1971). The specific molecular basis for most of these diseases is not yet understood; however, in many cases the basis of the disease has been determined to be a specific enzyme deficiency (McKusick, V. A., Ann Rev. Genet. 4:1 (1970)).
At present, no wholly acceptable method of gene therapy is known. Human genetic diseases are usually treated either by dietary therapy (such as the avoidance of phenylalanine by individuals who suffer from phenylketonuria), by drug therapy (such as the use of inhibitors of the enzyme xanthine oxidase (i.e., allopurinol), to reduce the accumulation of uric acid associated with gout and Lesch-Nyhan syndrome), or through gene product replacement therapy (such as by administering factor VIII to individuals who suffer from hemophilia).
Unfortunately, many genetic diseases do not yet respond to any of the above treatments. For example, genetic disorders of amino acid metabolism cannot generally be well controlled by dietary therapy. Storage diseases associated with lysosomal enzyme deficiencies have not thus far been found to respond to enzyme therapy. In addition, even where a disease may be controlled through any of the above methods, disease management is seldom perfect.
In response to the deficiencies of the above techniques, investigators have attempted to apply recombinant DNA technology to the treatment of genetic diseases. Such gene therapy can be broadly defined as a medical/surgical intervention in which the genome of the patient is purposely altered to ameliorate a pathophysiologic condition, and as such the term can be sub-divided into germ line and somatic cell gene therapy. Based on both ethical and practical criteria, it is not feasible to attempt germ-line gene therapy on human subjects. From an ethical perspective, modifying the germ line would change, albeit slightly, subsequent generations of humans, with the longterm effects not entirely predictable. Somatic cell gene therapy, in contrast, would only affect the individual subjected to the therapy, and the new gene would not enter the gene pool. From a practical perspective, germ line gene transfer is relatively inefficient, the fate of the injected genes cannot be predicted, and, perhaps most importantly, with few exception it would not be possible to determine the future phenotype (with respect to a given disease) of a single cell or early cleavage embryo.
Somatic cell gene therapy, however, does seem to be a reasonable approach to the treatment and cure of certain disorders in human beings. In a somatic cell gene delivery system, cells from the patient are removed, cultured in vitro, transfected, and reimplanted. Modifications of this basic scheme include, but are not limited to, choices of the cell type and cell donor (not necessarily the patient), the transfection protocol, and the site of reimplantation.
Several techniques have thus far been developed which offer promise as means for delivering DNA into an individual. A well-described technique involves retroviral vectors (Varmus, H., et al., in: RNA Tumor Viruses, Weiss et al., Eds. (Cold Spring Harbor Laboratory, New York, 1982); Varmus, H., Science 216:812 (1982); Risser, R., et al., Ann. Rev. Genet. 17:85 (1983)). In this approach, a particular gene is inserted into a retrovirus which is then introduced into an individual. Retroviruses store their genetic information in RNA, and, on entering a cell, reverse transcribe (hence the name “retro”) this information into DNA, which can then become integrated and expressed in the host cell's genome. In practice, a recombinant retrovirus containing the gene of interest and a portion of the retroviral genome (some retroviral genes are removed so that the virus cannot replicate) is constructed using genetic engineering methodologies. This artificial virus is utilized to infect marrow cells in vitro, and these cells are injected intravenously into lethally irradiated recipient mice, where they ultimately make their way to the marrow and spleen. Using this approach, genes encoding neomycin phosphotransferase (Williams, D. A., et al., Nature, 310:476–481 (1984)), adenosine deaminase (Williams, D. A., et al., Proc. Natl. Acad. Sc. USA, 83:2566–2570 (1986)), and hypoxanthine phosphoribosyltransferase (Miller, A. D. et al., Science 225:630–632 (1984)) have been expressed in mice.
In the past few years, it has become apparent that the implementation of retroviral based gene delivery systems in humans will face major obstacles, primarily related to properties of retroviruses themselves (Robertson, M., Nature 320:213–214 (1986), Marx, J. L., Science 232:824–825 (1986)). First, it has not been generally possible to achieve expression of mammalian genes in the retroviral vectors used to infect human cells, and until this problem is solved, the issue of regulated gene expression cannot be addressed. Second, when retroviruses are used to infect marrow cells in batch, essentially every cell is infected, and the site of retroviral integration into the host's genome varies from cell to cell. Since the infected cells are not characterized before reintroduction, the possibility of a deleterious intergration event cannot be eliminated. Third, as recombination between the replication-deficient retroviruses utilized for the infection and the endogenous retroviruses present in mammalian genomes is known to occur (Hock, R. A. et al., Nature 320:275–277 (1986)), there is the potential of initiating a chronic retroviral infection in the host animal. Fourth, marrow is probably not the optimal site of expression for many (if not most) genes of therapeutic import.
An alternative approach for gene therapy involves introducing DNA into a cell by chemical, as opposed to viral, techniques. In this approach, DNA is introduced into a recipient call by calcium phosphate-mediated transfection. In general, the recipient cells are first removed from an individual and incubated in the presence of a DNA solution containing the gene whose introduction is desired. After the gene has been introduced into the cell, the cell is returned to the individual. At present, the only cells which may be removed from an individual, treated, and subsequently reintroduced are bone marrow stem cells and skin fibroblasts (Anderson, W. F., Science 226:401–409 (1984)). Cline, M. J., et al. (Nature 284:422 (1982)) disclosed the successful transfer of a functional dihydrofolate reductase gene into the bone marrow of mice.
Present chemical techniques suffer from substantial drawback of low efficiency. Transfection has been found to occur in only one of 106 or 107 cells. Thus, since only approximately 107 or 108 cells may be routinely obtained from an individual by bone marrow transplantation, the chemical technique would mean that 10 to 100 stem cells would be transfected. In addition, the difficulty of culturing such cells for more than a few days is a substantial limitation to this method. It is currently believed that the presence of so few modified cells, when compared to the total number of cells in the bone marrow population, would have little therapeutic value.
A third major current approach to gene therapy involves the use of physical techniques such as micro-injection or electroporation. Microinjection involves the injection of DNA into isolated, individual cells. The technique, though extremely efficient, suffers from the disadvantage that only one cell at a time can be injected. This technique has been most successful in the introduction of DNA into fertilized mouse eggs (Gordon, J. W., et al., Science 214:1244 (1981); Wagner, E. F., et al., Proc. Natl. Acad. Sci. USA 78:5016 (1981); Wagner, T. E., et al., Proc. Natl. Acad. Sci. USA 78:6376 (1981); Palmiter, R. D., et al., Nature 300:611 (1982)). Hammer, R. E., et al., (Nature 311:65 (1984)), used this technique to partially correct a mouse with a defect in its growth hormone production. Electroporation involves the transport of DNA directly across a cell membrane through the use of an electric current. It has been used to transfer DNA into B lymphocytes (Neumann, E., et al., EMBO J. 1:841 (1982)).
In summary, various conventional and recombinant techniques have been proposed for the treatment of genetic diseases. At present, however, no single technique appears to be wholly satisfactory. The use of viral vectors suffers from their potential for rearrangement of endogenous genes, as well as their potential for inducing carcinogenesis. Physical techniques, though highly efficient, are at present incapable of application to the large numbers of cells which would need to be transfected in order to provide a reasonable therapy. Chemical procedures involve the introduction of DNA into a cell which had previously been extracted from a subject individual. At present, the technique is limited to bone marrow cells and fibroblasts, and is not efficient enough to constitute a viable therapy. The state of this field is reviewed by Friedman, T., et al. (Science 175:949–955) (1972) and Anderson, W. F. (Science 226:401–409 (1984)).
Binding proteins, such as antibodies, are widely used to assay for the presence or concentration of particular molecules, such as antigens or haptens. An antigen is a molecule which, when introduced into an animal, provokes the animal to produce antibodies which are capable of binding to it. In contrast, a hapten molecule is capable of binding to antibodies, but is incapable of eliciting their production. Antibodies bind to both haptens and antigens by identifying particular structural regions (known as “epitopes”) of the molecules. A hapten or antigen molecule may contain more than one epitope region.
Preparations of antibodies may be broadly divided into two classes. Polyclonal antibody preparations are obtained by injecting (or otherwise presenting) an antigen molecule into an animal. The presence of the antigen molecule stimulates antibody-producing cells to produce species of antibodies capable of binding to the epitopes of the antigen. Different antibody-producing cells are capable of producing different antibody molecules. Thus, the introduction of an antigen into an animal results in the production of an array of different antibody molecules which includes antibodies capable of binding to each of the epitopes of the antigen molecule. Because such a preparation includes antibodies which were produced from different producer cells, it is termed a “polyclonal” antibody preparation.
Excellent reviews of the methods and techniques for preparing polyclonal antibodies can be found in Microbiology, 2nd Edition; Davis, B. D., et al.; Harper & Row, New York (1973), pp. 352–358; and Remington's Pharmaceutical Sciences, 16th Edition, Osol, A., Ed., Mack Publishing, Easton, Pa. (1980), pp. 1315–1351.
The fact that individual antibody-producing cells are capable of producing only a single species of antibody is highly significant. Such individual cells can be clonally purified and fused to immortalized myeloma cells, thereby producing an immortalized cell which is capable of producing a single antibody species. Such fusion cells are known as “hybridoma” cells, and the antibodies which they produce are known as “monoclonal” antibodies. The procedures for producing monoclonal antibodies are disclosed in U.S. Pat. No. 4,172,124 (Koprowski, H., et al.) and in Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennett, R. H., et al. (Eds.), Plenum Press, NY (1980).
The distinction between monoclonal and polyclonal antibodies does not lie in their individual specificity or binding affinity. Both types of antibody molecules exhibit equivalent specificity and binding affinity toward antigen molecules. Preparations of polyclonal antibodies comprise either an unfractionated mixture, or purified mixtures of IgG molecules, of different antibody species, many of which are capable of binding to different epitopes. In contrast, a preparation of monoclonal antibodies contains only a single antibody species. Because they are composed of a single species, preparations of monoclonal antibodies (though not necessarily the antibodies themselves) may possess greater specificity than can a preparation of polyclonal antibodies.
Significantly, in order to produce either monoclonal or polyclonal antibodies, it is generally necessary for one to present substantial amounts of the antigen to the antibody-producing cells. Thus, in general, it is necessary to isolate and purify the antigen molecules before antibody production can be induced. In practice, however, it is often difficult to isolate and purify particular antigen molecules. This is especially true if the antigen molecules are hormones, membrane proteins, or other molecules which are present at only very low concentrations in source materials. Thus, for such molecules, it is often difficult or not possible to obtain antibodies. Hence, a need exists for a method of producing antibodies which does not require the prior isolation or purification of the antigen molecule.